High glucose mediates aberrant redox and tight junction protein expression in APRE-19.
ARPE- 19 cells were maintained in DMEM/F-12 medium containing 2% serum with antibiotics containing 5.5mM glucose (LG) or 25mM glucose (HG) for 5 days like those described previously (33). Western blotting and QPCR detected protein and mRNA levels. Data are presented as SEM+/−SE and p value of <0.05 is considered significant when compared between LG and HG using Student’s t-test.
(A-B) TXNIP level is increased in HG compared to LG (p<0.04) while that SOD1 and Trx1 are down regulated significantly. Tubulin is used as a control protein.
(C-E) Protein and mRNA levels of ZO-1 also decrease in HG compared to LG.
(F) Furthermore, ZO-1 immunostaining (red) is also disrupted at the plasma membrane under HG (yellow arrows) when compared to LG. Furthermore, addition of 2μM azaserine (an inhibitor of TXNIP, ref. 25) in the last 24h of the experiment restores ZO-1 plasma membrane staining in HG. A representative of n=3 is shown here.